The translocation assembly module (TAM) catalyzes the assembly of bacterial outer membrane proteins .

The bacterial translocation assembly module (TAM) contains an outer membrane protein (OMP) (TamA) and an elongated periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). TAM has been proposed to play a critical role in the assembly of a small subset of OMPs produced by Proteobacteria based on experiments conducted using and/or deletion or mutant strains and using biophysical methods. Recent genetic experiments, however, have strongly suggested that TAM promotes phospholipid homeostasis.

The translocation assembly module (TAM) catalyzes the assembly of bacterial outer membrane proteins in vitro.

The translocation and assembly module (TAM) has been proposed to play a crucial role in the assembly of a small subset of outer membrane proteins (OMPs) in Proteobacteria based on experiments conducted in vivo using tamA and tamB mutant strains and in vitro using biophysical methods. TAM consists of an OMP (TamA) and a periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). Here we examine the function of the purified E.

OPA1 helical structures give perspective to mitochondrial dysfunction.

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria. Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes.

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus.

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted.

Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact.

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place.

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions.

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb, a structural and functional surrogate of Ca, to generate the protein states with well-defined protein-metal ion stoichiometry.

Phosphatidylinositol 4,5 Bisphosphate Controls the cis and trans Interactions of Synaptotagmin 1.

Synaptotagmin 1 acts as the Ca sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here, we describe measurements on full-length membrane-reconstituted synaptotagmin 1 using site-directed spin labeling in which we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate, even if phosphatidylserine (PS) is present in the cis membrane.

The Fluidity of Phosphocholine and Maltoside Micelles and the Effect of CHAPS.

The dynamics of phosphocholine and maltoside micelles, detergents frequently used for membrane protein structure determination, were investigated using electron paramagnetic resonance of spin probes doped into the micelles. Specifically, phosphocholines are frequently used detergents in NMR studies, and maltosides are frequently used in x-ray crystallography structure determination. Beyond the structural and electrostatic differences, this study aimed to determine whether there are differences in the local chain dynamics (i.

A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis.

The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane.

Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1.

Synaptotagmin-1 (Syt1) functions as the Ca sensor in neuronal exocytosis, and it is routinely incorporated into lipid bilayers along with other components of the fusion machinery in order to reconstruct the in vivo fusion process. Here, we demonstrate that the detergent used to reconstitute full-length Syt1 has a significant effect on the state of the protein in bilayers. When octyl-β-d-glucopyranoside is used to reconstitute the protein, Syt1 is present in an aggregated state that is mediated by the long juxta-membrane linker.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca-dependent manner. In these cases, membrane association is triggered by Ca binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd, in lieu of Ca to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding.